A multivariate retrospective analysis of high‐grade gliomas: Survival and prognostic factors

Abstract Objectives High‐grade gliomas (HGGs) are highly malignant, aggressive, and have a high incidence and mortality rate. The aim of this study was to investigate survival outcomes and prognostic factors in patients with HGGs. Methods In this retrospective study, a total of 159 patients with histologically confirmed HGGs were included. The recruitment period was from January 2011 to December 2019. We evaluated patient demographic data, tumor characteristics, treatment methods, immunocytochemistry results, overall survival (OS) time, and progression‐free survival (PFS) time using Kaplan–<>Meier survival analysis with log‐rank testing. Additionally, we employed Cox regression analysis to identify independent factors associated with survival outcomes. Results Kaplan–Meier survival analysis revealed that the 1‐, 2‐, and 5‐years OS rates were 81.8%, 50.3%, and 12.6%, respectively. Similarly, the 1‐, 2‐, and 5‐years PFS rates were 50.9%, 22.4%, and 3.1%, respectively. The median OS duration was 35.0 months. The univariate analysis indicated that postoperative pathological classification, grade, and age were significantly associated with patient outcomes (p < 0.01). Among the patients, 147 received concurrent chemoradiotherapy, while 12 did not. The immunohistochemical markers of ki‐67, MGMT, IDH1R132H, and p53 demonstrated statistically significant differences in their prognostic impact (p = 0.001, p = 0.020, p = 0.003, and p = 0.021, respectively). In conclusion, we found that grades, age, pathological classification, ki‐67, MGMT, and IDH1R132H expression were statistically significantly associated with PFS (p < 0.01, p = 0.004, p = 0.003, p = 0.001, p = 0.036, and p = 0.028). Additionally, immunohistochemical expressions of TRIB3 and AURKA were significantly higher in patients with shorter survival (p = 0.015 and p = 0.023). Conclusions Tumor grade and the use of concurrent chemoradiotherapy after surgery were independent prognostic factors that significantly influenced patient survival. Additionally, tumor grade and MGMT expression were found to be independent factors affecting progression‐free survival (PFS). Notably, the expression of TRIB3 and AURKA was higher in patients with poor survival outcomes.


| BACKGROUND
Gliomas, which derive from the progenitor cells or neuroglial stem, are responsible for the majority of deaths from primary brain tumors. 1 The World Health Organization (WHO) classification of gliomas places gliomas into grades 1-4, with grades 1 and 2 indicating low-grade gliomas and grades 3 and 4 high-grade gliomas (HGGs).HGGs are highly malignant and invasive, with high morbidity and mortality.Surgical resection is the conventional treatment for HGGs, but it is difficult to completely remove clean because the boundary between normal tissue and tumor cells is not clear and tumor cells are highly infiltrative and invasive. 2Therefore, it is necessary to treat HGGs with adjuvant postoperative chemoradiotherapy as soon as possible.Current NCCN guidelines propose that surgical resection, concurrent chemoradiotherapy, and adjuvant temozolomide (TMZ) as a standard treatment for HGGs. 3 Classic research has shown that radiotherapy in combination with chemotherapy, followed by up to six cycles of adjuvant TMZ, significantly boosted 5-year overall survival (OS) and progression-free survival (PFS) compared to radiotherapy alone. 46][7][8] In addition to standard therapy, the research of targeted therapy, immunotherapy, and tumor-treating fields therapy is also advancing.Some biological markers suggest that gliomas may have more drug options, such as IDH mutation, EGFR amplification, and BRAF V600E mutations.Ivosidenib, IDH1/2 inhibitors, had a higher median PFS of 13.6 months for recurrent IDH-mutated high-grade gliomas. 9The median survival time of anti-EGFR antibody nimotuzumab combined with radiotherapy in the treatment of newly diagnosed HGGs was 17.8 months. 10For recurrent gliomas with BRAF V600E mutation, trametinib combined with dabraafenib can be used.More biomarker may herald a more targeted drugs, this is also helpful for the treatment of HGGs.
HGGs carry a dismal prognosis and exhibit a high recurrence rate, emphasizing the crucial importance of investigating prognostic factors that impact glioma survival.In this study, 159 patients with HGGs were selected and the association of relevant factors including biomarkers with prognosis was evaluated.The objective of this study is to ascertain the factors that influence the OS and PFS of patients with HGGs, thereby aiding in the exploration of advanced treatment strategies.

| Patients
This study retrospectively analyzed 159 HGGs patients treated in the first hospital of Jilin University from January 2011 to December 2019.All patients were newly diagnosed as grade III-IV (WHO) glioma, and excluded if they were pregnant or lactating women, or had other brain tumors.The ethics committee of the First Hospital of Jilin University has reviewed and approved this study, with the ethical number of 2022-KS-010.

| Immunochemical staining and analyses
Among 159 patients, 20 glioblastoma patients with the longest survival periods and 20 glioblastoma patients with the shortest survival periods were selected, and those with unresected surgery and without concurrent chemoradiotherapy and at least four cycles of chemotherapy were excluded.The postoperative pathological tissues of this 40 patients were stained by IHC staining of Tribbles 3 (TRIB3) and Aurora kinase A (AURKA).IHC staining was performed as previously reported.Primary antibodies included TRIB3 (rabbit monoclonal, 1:100 dilution, #ab75846, Abcam, Cambridge, UK) and AURKA (rabbit polyclonal, 1:100 dilution, #ab52974, Abcam, Cambridge, UK).The secondary antibody was purchased from SeraCare (goat anti-rabbit, #5220-0336, SeraCare, Massachusetts, USA).The formula number of pixels in a zone multiplied by score of the zone divided by total number of pixels in the zone was used to calculate the IHC scores of each patient by ImageJ software, and the scores were statistically analyzed.

| Therapy
All patients received standard treatment, including maximum tumor resection, postoperative concomitant chemoradiotherapy, and sequential chemotherapy.Radiotherapy modality was generally chosen as intensity-modulated radiation therapy (IMRT) or VMAT.The target area was outlined according to preoperative and postoperative CT and MRI.The median radiation dose was 60 Gy (12-66 Gy).The chemotherapy regimen was usually oral TMZ during radiotherapy, and took it 4 weeks after radiotherapy, generally lasting for more than half a year.

| Statistical analysis
SPSS 21.0 (SPSS Inc., Chicago IL, USA) was used for statistical analysis, and the qualitative data were expressed using rate.OS was estimated from the date of diagnosis to the date of death.PFS was estimated from diagnosis to disease progression or death or the date of last followup.Kaplan-Meier method were used to estimate OS and PFS among different factors and log-rank method was used to test the differences in survival time distributions.The statistically different factors observed in the log rank method were incorporated into the Cox regression model using the forward: LR method to evaluate the independent risk factors affecting the prognosis of HGGs.The statistical significance of differences was set at P < 0.05.48.4% of patients received radiation doses between 54 and 60 Gy, and 36.5% of patients received doses more than 60 Gy.Among these patients, 44 patients were given radiotherapy less than 4 weeks after operation, while most radiotherapy (n = 100, 62.9%) started more than 4 weeks after section.Tumor location was as followed: right hemisphere (n = 70, 44.0%), left hemisphere (n = 81, 50.9%) and other sites (n = 8, 5.0%).Around 63.5% of the tumors were single lesion, and the remainder were diffuse multiple lesions.Expressions of ki-67, IDH1 R132H, MGMT, p53, and EGFR were detected by IHC, and the results are reported in Table 1.
Immunohistochemical results showed that high expression of ki67, low expression of MGMT, negative IDH, and positive TP53 were significantly correlated with longer OS and PFS.

| Prognostic factors in multivariate analysis
The results of multivariate analysis in OS are displayed in Table 2.The multivariate survival analysis identified the following parameters as independent factors associated with prolonged OS time: the grade of gliomas (P = 0.017, HR 2.553, 95%CI 1.2-5.5)and patients received radiotherapy (P = 0.010, HR 3.539, 95%CI 1.4-9.3).
Our analysis reveals that patients with pathological of grade IV and those who did not receive postoperative radiotherapy are at higher risk of mortality.More noteworthy, high expression of MGMT in immunohistochemistry and GBM were correlation to tumor progression (Figure 4A-H). 3.5 | High expression of TRIB3 and AURKA was associated with poor survival IHC staining showed that the expression of TRIB3 and AURKA was significantly increased in patients with short survival (Figure 5A,B,D,E).The short survival group had a significantly higher TRIB3 score than the long survival group (24.5 vs. 15.2,P = 0.015), and the AURKA also exhibits a similar phenomenon (21.9 vs. 13.7,P = 0.023), respectively (Figure 5C,F).

| DISCUSSION
HGGs are among the most malignant tumors with high aggressiveness and heterogeneity.The prognostic factors and treatment of HGGs are complex and changeable.Therefore, the formulation of individualized treatment plan according to the prognostic factors can guide clinical work and improve the treatment effect.Surgical resection is the most important treatment for glioma, and the maximum resection of the tumor may improve the survival of patients.A meta-analysis showed significantly lower mortality with GTR compared with STR.Any resection, as compared with biopsy alone, similarly reduced the risk of death at 1 and 2 years. 12Large the scope of extended resection is associated with more survival benefit for HGGs.A study revealed that independent predictors of postoperative epilepsy included temporal lobe involvement, preoperative glioma-related epilepsy, lower WHO grade and non-gross-total resection. 13For recurrent HGGs, re-resection or not had no significant effect on median OS. 14 Although our results suggested that the surgical method did not provide a statistically significant prolongation survival time.This may be attributed to the limited number of patients with partial resection and simple biopsy, and the statistician may have data bias.Although no statistical differences were observed, the median OS without surgery, partial resection, and total resection were 13.0 months, 25.0 months, and 36.0 months, respectively.It can be seen that total resection still has a better prognosis compared with the other two groups.
An epidemiological statistic revealed that men were more prone to gliomas than women. 15Our study did not indicate there have significant difference in OS and PFS between males and females.However, both median OS (37.0 months vs. 32.0months) and median PFS (14.0 months vs. 12.0 months) were higher in females than males.Oligodendroglioma occurred predominantly in the median age of 43 years old, diffuse astrocytoma induced at the median age of around 48 years old, anaplastic oligodendroglioma developed at the median age of 49, anaplastic astrocytoma occurred at around 53 years old, whereas GBM peak in the median age of 64. 16We revealed that the median ages of oligodendroglioma, astrocytoma, anaplastic oligodendroglioma, anaplastic astrocytoma, and GBM were 46.0, 44.0, 42.0, 45.0, and 55.5 years old.These results suggested that HGGs mainly occurred in 40-60 years old.Our findings revealed that patients younger than 40 years old had significantly better OS and PFS than patients in the other ages(P < 0.001 and P = 0.004).
Radiotherapy is an essential therapeutic method for HGGs patients.A clinical trial found no statistically significant difference in survival rate in the dose range of 60-80 Gy 17 ; however, another study found that patients received 80 Gy and 90G y had significantly longer OS than patients received 60 Gy. 18In our study, no significant difference in OS or PFS was found between radiotherapy doses, and patients who received doses more than 60 Gy had the highest median OS (36.0 months vs. 32.0months) but the lowest median PFS (12.0 months vs. 13.0 months).It is difficult for conventional radiotherapy technology to carry out radical treatment for tumors close to dangerous organs.Patients receive particles, such as protons or carbon ions, and this modality with higher linear energy transfer can improve the dose distribution, induces DNA double strand breaks, killing cell more efficiency, which can ameliorate the curative effect of radiotherapy.The utilization of radiation modalities that promote enhanced local dose deposition, escalate irradiation dosage for the biological target volume, and enable intraoperative radiotherapy may represent the prospective developmental trajectory for high-grade glioma radiotherapy in the future.In our study, we use are x-ray radiation, compared with no postoperative radiotherapy, postoperative conventional radiotherapy can improve the median OS of HGGs patients significantly, which is consistent with the conclusion of our study (P = 0.003).IDH1 mutation at codon 132 is related to the prognosis of HGGs.Patients with IDH mutant glioblastoma usually have a better prognosis than patients with IDH wild-type glioblastoma.Ivosidenib had a higher median PFS of 13.6 months for recurrent IDH-mutated high-grade gliomas. 10However, the CNS WHO 5 has classified IDHmutant gliomas into oligodendroglioma and astrocytoma, and glioblastomas are all IDH wild type, so glioblastomas F I G U R E 1 Kaplan-Meier analysis showed that age of onset, tumor type, KPS score, WHO grade, postoperative concurrent chemoradiotherapy, and postoperative radiotherapy were significantly associated with overall survival in high-grade glioma patients.cannot benefit from ivosidenib, but for oligodendroglioma and astrocytoma, this is undoubtedly a breakthrough in the treatment.
MGMT promoter methylation is a biomarker to predict the benefit from TMZ chemotherapy.The gold standard examination for MGMT methylation is polymerase chain reaction (PCR), but for some cases where the examination conditions are limited or PCR cannot be performed due to personal reasons, it can also be replaced by immunohistochemistry (IHC).Anda et al. 19 found that the median PFS was 9.0 months and the median OS was 12.0 months in patients of high MGMT expression, while the median PFS was 15.0 months and the median OS was 22.0 months in patients of low MGMT expression.High MGMT expression is associated with poor prognosis.This is consistent with our research conclusions.Among the enrolled patients, 70 patients had high MGMT expression and 42 patients had low MGMT expression, except for patients with unknown MGMT expression.The median OS of low and high MGMT expression were 44.0 and 26.0 months, respectively (P = 0.020), median PFS were 15.0 and 10.0 months, respectively (P = 0.036).
P53 is a tumor-suppressor gene involved in the occurrence of HGGs.As a highly specific marker, P53 is highly positive in HGGs, and the positive rate is higher than that of patients with low-grade gliomas.Overexpression of P53 suggests that the prognosis of patients is poor.In our study, p53-positive indicated poor prognosis.Compared with negative, there was a significant difference in median OS (P = 0.021), but no significant difference in median PFS (P = 0.070).This result was consistent with a retrospective analysis of anaplastic gliomas. 20-67 is a nuclear protein that describes the proliferative stage of the cell cycle and reflects the proliferative activity of HGGs cells.It is an independent prognostic factor in gliomas, and a higher expression content is associated with a worse prognosis. 21In our study, the median PFS of patients with ki-67 ≥ 15 and those with ki-67 < 15 were 12.0 and 23.0 months, respectively (P = 0.001).The median OS of patients with ki-67 ≥ 15 was 26.0 months, while those with Ki-67 < 15 had not yet reached (P = 0.001).
We examined several biomarkers that are not routinely used in glioma IHC detection and found that TRIB3 and AURKA were associated with poor prognosis of the disease.Multiple studies have shown that TRIB3 expression is upregulated in response to various stressors, including oxidative stress, metabolic stress, as well as endoplasmic reticulum (ER) stress. 22Recently, several studies have identified TRIB3 as a crucial regulator in tumorigenesis and tumor progression.TRIB3 is overexpressed in various cancer tissues and is closely associated with poor prognosis in patients, including breast cancer, 23 and colorectal cancer, 24 lung cancer. 25Unfortunately, the precise roles of TRIB3 in high-grade gliomas (HGGs) remain elusive.The mRNA expression of TRIB3 in glioblastoma clinical samples was higher than that in normal brain tissue. 26TRIB3 is also expected to be a new target for glioma treatment.In our study, we selected 40 patients with glioblastoma according to different prognosis.Through IHC analysis of these patients, we found the expression of TRIB3 in the short-term survival was significantly higher than those in the long-term survival.This difference was verified by ImageJ software analysis (P = 0.015).
AURKA is a serine/threonine kinase that regulates cellular mitosis and is located on chromosome 20q13.2. 27I G U R E 2 Kaplan-Meier analysis showed that ki67, IDH1R132, MGMT, and P53 were significantly associated with overall survival in high-grade glioma patients.
| 9  As a cell cycle regulator, AURKA overexpression leads to centrosome expansion and aneuploidy.AURKA promotes tumorigenesis by participating in epithelial-mesenchymal transformation, proliferation and metastasis of cancer cells, apoptosis, and self-renewal of stem cells. 28In glioma, inhibition of AURKA expression can inhibit the growth of tumor cells, which can be used as a potential therapeutic target.Similar to TRIB3, we also found that the expression of AURKA in the short survival was significantly higher (P = 0.023).It is anticipated that further investigation into TRIB3 and AURKA could lead to the development of novel therapeutic targets for glioma, such as MGMT and EGFR.Reviewing our data, the overall OS and PFS were higher than the guidelines, analysis may be related to the following factors.Most patients received multidisciplinary consultation, and this mode of treatment can make the treatment of patients more standardized and timely.A subset of patients received longer cycles of TMZ chemotherapy.The longest surviving patient with glioblastoma was 86 months, and this patient received TMZ for a total of 80 months.Long cycles of temozolomide have also been reported to improve survival. 29Some patients who have survived for a long time have a pathological type of astrocytoma or oligodendroglioma, which has a better prognosis.Moreover, because of lack of the molecular pathology F I G U R E 5 (A, B) Immunochemical staining indicated that the expression of TRIB3 was significantly higher in patients with short survival than in patients with long survival.(C) The TRIB3 immunohistochemical score of the long survival group was significantly higher than that of the short survival group.(D, E) Immunochemical staining indicated that the expression of AURKA was significantly higher in patients with short survival than in patients with long survival.(F) The AURKA immunohistochemical score of the long survival group was significantly higher than that of the short survival group.*P < 0.05.information in our samples, which may cause some statistical bias, such as the prognosis of grade IV patient with IDH mutated is significantly better than that of grade IV patients with IDH wild type, two different subtypes of gliomas may have different prognostic factors affecting OS and PFS, which is an underexplored part of our study.
In conclusion, this is a meaningful retrospective study with considerable number of HGGs patients, and we did a full-scale analysis.Age, the grade and histology of tumor, preoperative KPS score, radiotherapy, concurrent chemotherapy, the expression of p53, MGMT, IDH1 R132H, and ki-67 correlate significantly with the survival prognosis of these patients with HGGs.The high expression of TRIB3 and AURKA immunohistochemistry may indicate a shorter survival time of patients.Through this study, we can provide evidence for judging HGGs patients prognosis and a direction for the exploration of HGGs treatment.

F
I G U R E 4 (A-D) The immunohistochemical expression of ki67, IDH, MGMT, and P53 in patients with long survival.(E-H) The immunohistochemical expression of ki67, IDH, MGMT, and P53 in patients with short survival.
Clinical characteristics of 159 high-grade gliomas.
fused radiotherapy at the initial diagnosis of HGGs because of physical intolerance or personal reasons.15.1% of patients received radiation doses no more than 54 Gy,T A B L E 1 Variables related to overall survival in univariate analysis and mulvariate Cox analysis.
T A B L E 2

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Variables related to progress-free survival in univariate analysis and mulvariate Cox analysis.